Comprehensive stability evaluation of honey sugars in a candidate certified reference material using integrated statistical and metrological approaches

Abstract The development of matrix-matched certified reference materials (CRMs) for honey is essential to ensure analytical reliability in quality control and authenticity testing. A fundamental requirement for CRM certification is a comprehensive understanding of its stability, particularly with respect to sugar components that are prone to degradation. This study systematically evaluated the short-term stability of fructose, glucose, and sucrose in a candidate honey CRM under controlled storage conditions (2 °C, 4 °C, and −10 °C) over a 3-month period, in accordance with ISO 35 guidelines. Sugar concentrations were quantified using high-performance liquid chromatography with refractive index detection (HPLC-RID) method. Stability was assessed using a tiered statistical and metrological framework. Initial descriptive statistics and ordinary least squares regression revealed robust stability for fructose and glucose while non-linear degradation for sucrose at 25 °C was observed. This was confirmed by t-tests, which pinpointed significant sucrose degradation after the first month. A two-way ANOVA validated a significant time–temperature interaction effect (p < .001) for sucrose, demonstrating that its stability is governed by storage temperature. The relative discrepancy (RD) metric quantified the instability, with a marked increase (from 1.73% to 36.94%) in the RD of sucrose at 25 °C, confirming severe instability. The definitive assessment used weighted least squares regression to calculate the uncertainty of the certified value in terms of short-term stability (usts). The results confirmed a highly significant degradation trend for sucrose at 25 °C (p = .004) and yielded a high usts value of 2.039, quantifying its severe lability. Refrigeration at 4 °C was identified as the optimal condition, effectively stabilising the entire sugar profile and yielding the lowest combined uncertainty (e.g., sucrose usts = 0.294). The current study provides the critical metrological evidence required for CRM certification and establishes that refrigeration at 4 °C is essential to preserve the integrity of the honey matrix for use as a reference material.